How to increase tritium counting efficiency?

Wolfgang Schechinger Wolfgang.Schechinger at med.uni-tuebingen.de
Tue Mar 21 07:50:42 EST 2000


Juan, 

you could check if some 100ul of 2% SDS and .3M NaOH dissolve the DNA 
from the filter, once the filter has been placed inside the 
scintillation tube. Vigorous agitation could be necessary. The paper 
maybe will be dissolved, too. A higher concentration of NaOH could be 
desirable, but you might have to check if your scintillation cocktail 
will like it; maybe you'll then have to neutralize after dissolving.

I have done experiments where cells were incubated with media 
containing 3H thymidine. Cells then were fixed with trichloroacetic 
acid, extensively washed and dissolved with the reagent above 
(30 min, additional pipetting). So I'd assume that you could 
be able to dissolve the DNA on your filter that way.

A (non radioactive) alternative would be the application of the BrdU 
incorporation method (also as kit available from Roche Diagnostics, 
you might contact them for discussing details) if your polymerase 
accepts BrdU (you have a bromine instead of the methyl group). 
Detection then would be using a peroxidase conjugated antibody 
against BrdU ending up in a colorimetric assay.

All the best, 

Wolfgang


> From:          jjmirujo at unav.es (" J. Martinez-Irujo")
> Subject:       How to increase tritium counting efficiency?
> Date:          21 Mar 2000 10:50:52 -0000
> Organization:  Universidad de Navarra
> X-To:          "methods at net.bio.net" <methods at hgmp.mrc.ac.uk>
> To:            methods at hgmp.mrc.ac.uk

> Dear Netters,
> We are measuring DNA polymerase activity following the incorporation
> of tritium labeled nucleotides into DNA. DNA is then bound to DE-81
> paper, washed and radioactivity measured by liquid scintillation. We
> have noticed that c.p.m. of paper bound tritium are about 5-fold
> lesser than c.p.m. of soluble nucleotides. Anybody knows a method to
> increase counting efficiency of paper bound DNA? (solubilizing the
> sample, perhaps). Any suggestion will be appreciated.
> 
> --
> Juan J. Martinez Irujo
> Departamento de Bioquimica
> Universidad de Navarra
> Pamplona, Spain
> 
> 
> ---
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Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
University of Tuebingen, Germany
email: wolfgang.schechinger at med.uni-tuebingen.de 
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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